Deep UV resonant Raman spectroscopy for photodamage characterization in cells.

Abstract

We employed deep UV (DUV) Raman spectroscopy for characterization of molecular photodamage in cells. 244 nm light excitation Raman spectra were measured for HeLa cells exposed to the excitation light for different durations. In the spectra obtained with the shortest exposure duration (0.25 sec at 16 µW/µm(2) irradiation), characteristic resonant Raman bands of adenine and guanine at 1483 cm(-1) and tryptophan and tyrosine at 1618 cm(-1) were clearly visible. With increasing exposure duration (up to 12.5 sec), these biomolecular Raman bands diminished, while a photoproduct Raman band at 1611 cm(-1) grew. By exponential function fitting analyses, intensities of these characteristic three bands were correlated with sample exposure duration at different intensities of excitation light. We then suggest practical excitation conditions effective for DUV Raman observation of cells without photodamage-related spectral distortion.

Full text

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Deep UV resonant Raman spectroscopy for
photodamage characterization in cells
Yasuaki Kumamoto,1,2 Atsushi Taguchi,2 Nicholas Isaac Smith,3
and Satoshi Kawata1,2,*
1Department of Applied Physics, Graduate School of Engineering, 2-1 Yamadaoka, Suita,
Osaka 565-0871, Japan
2Nanophotonics Laboratory, RIKEN Advanced Science Institute, 2-1 Hirosawa, Wako Saitama 351-0198, Japan
3Biophotonics Laboratory, Immunology Frontier Research Center, Osaka University, 2-1 Yamadaoka, Suita,
Osaka 565-0871, Japan
*kawata@ap.eng.osaka-u.ac.jp
Abstract: We employed deep UV (DUV) Raman spectroscopy for
characterization of molecular photodamage in cells. 244 nm light
excitation Raman spectra were measured for HeLa cells exposed to the
excitation light for different durations. In the spectra obtained with the
shortest exposure duration (0.25 sec at 16 µW/µm2 irradiation),
characteristic resonant Raman bands of adenine and guanine at 1483
cm1 and tryptophan and tyrosine at 1618 cm1 were clearly visible.
With increasing exposure duration (up to 12.5 sec), these biomolecular
Raman bands diminished, while a photoproduct Raman band at 1611
cm1 grew. By exponential function fitting analyses, intensities of these
characteristic three bands were correlated with sample exposure
duration at different intensities of excitation light. We then suggest
practical excitation conditions effective for DUV Raman observation of
cells without photodamage-related spectral distortion.
©2011 Optical Society of America
OCIS codes: (300.6450) Spectroscopy, Raman; (300.6540) Spectroscopy, ultraviolet;
(350.1820) Damage; (170.1530) Cell analysis; (170.5660) Raman spectroscopy.
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1. Introduction
Deep UV (DUV) Raman spectroscopy selectively visualizes nucleotide bases and
aromatic amino acids in cells due to the resonant effect [1–8]. In the resonant Raman
condition, where the electronic transition energy of a molecule corresponds to photon
energy of Raman excitation light, the Raman scattering intensity of the molecule is
enhanced by as much as 106 compared to the non-resonant Raman scattering [9]. Because
other biological compounds in cells, such as lipids and sugars, are not in the resonant
condition at DUV, Raman scattering from nucleotide bases [1–7] and aromatic amino
acids [1–8] are selectively enhanced in DUV resonant Raman spectroscopy of cells.
In DUV Raman spectroscopy of cells, molecular photodegradation often occurs
during the measurement [10]. The photodamage of biomolecules is caused via strong
absorption. The photon energy of DUV is sufficiently high for excited molecules to be
broken or react with surrounding media [11–13]. Particularly, the accompanying
photodamage is a serious issue for observation of biomolecules in a small volume [14].
This makes it difficult to achieve microscopic observation of living cells with high spatial
resolution [15,16]. It is then important to understand the molecular degradation in cells
during DUV Raman measurement, and to be aware of the light intensities and exposure
times which bring about such effects.
DUV-induced molecular degradation in whole cells has not previously been
characterized. Degradation [17,18] or phototransient states [19] of some individual
biomolecules have been characterized by using DUV Raman spectroscopy, but individual
molecules don’t accurately represent the photodamage that occurs in whole cells.
Molecular level analyses of the ensemble of molecules are required for useful
characterization of photodamage in whole cells.
In this study, we employed DUV resonant Raman spectroscopy for direct
characterization of the accompanying molecular degradation in cells. The characteristic
resonant Raman bands of nucleotide bases and aromatic amino acids were analyzed in the
DUV Raman spectra of HeLa cells exposed to excitation light for different durations at
different intensities. The intensity of characteristic Raman bands were correlated with the
exposure durations at different excitation intensities. In the obtained spectra, we identified
the growth of a photoproduct band. The growth of the photoproduct band was also
correlated with the exposure durations of excitation light at different intensities. This
study will suggest an exposure condition effective for practical use of DUV resonant
Raman spectroscopy of cells without spectral distortion as well serve to provide
quantitative understanding of the Raman spectra of cells, which are often composed of
both intact sample signals and photoproduct noise.
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2. Materials and methods
2.1. Deep UV Raman spectroscopy
A purpose-built Raman microscope was used for the experiments. The 244 nm continuous
oscillation line of an Argon ion SHG laser (Coherent Innova 300C MoToFRED) was used
as the Raman excitation light. 244 nm is the wavelength optimized to resonantly excite
both nucleotides and aromatic amino acids. The laser intensity is controlled by an ND
filter. The expanded and collimated beam was introduced into a 40x UV achromatic
objective lens (OFR LMU-40x UVB, NA = 0.5) equipped on a Nikon inverted
microscope via non-polarized beam splitter. The beam was focused at the interface
between the sample and the substrate. The area of focus was estimated to be ~1 µm2. The
scattering light was collected by the same objective with back scattering geometry.
Rayleigh scattering light was rejected by longwave-pass edge filter having an optical
density of 4.0. The transmitted light was guided into a spectrometer and dispersed by a
3600 G/mm grating. Raman spectra were measured using a back-illuminated cooled-CCD
(Princeton Instruments SPEC-10 2KBUV). The grating dispersion was calibrated using
the excitation wavelength, and the Raman bands of sulfate ion (981 cm1) and acetonitrile
(2249 cm1) [20]. All Raman measurements were done at room temperature.
Raman spectra were measured at different sample exposure durations. For a fair
spectral comparison among different sample exposure durations, we measured the same
sample area repeatedly with a constant spectral acquisition time and then observed how
the spectra changed with accumulating exposure. Each spectrum was obtained by
integrating the spectral signal on the detector over a large number (200) of micro-
observation positions in order to obtain sufficient S/N for quantitatively analyzing slight
changes in cell spectra. Measurement points were separated from each other in space by 5
µm, a distance 5 times larger than the focal spot diameter. Since the output spectrum was
accumulated from 200 positions, the final data structure is just one spectrum for each
exposure time. The entire measurements were then repeated 3 times to provide a measure
of repeatability. The standard deviations shown in Figs. 1, 2 and 3 are therefore calculated
from the deviations between the three output spectra for the same conditions.
2.2. Samples
HeLa cells were purchased from Genlantis, Inc. The cells were cultured on quartz
substrates with Dulbecco’s modified Eagled medium for 2~4 days in a 5% CO2 incubator
at 37 degrees and 100% humidity. The cells were used at the confluent density.
Different treatments were performed on cultured cells according to the purpose of the
Raman measurements. For experiments done to quantitatively correlate the molecular
degradations with the exposure durations and intensities, desiccated cells were used,
because desiccated cells are not affected by diffusion of molecules. The desiccation
treatment of the cells was performed according to the literature [21]. Briefly, the
procedure was to rinse the cells with Tyrode’s solution, then the cells were placed in a
desiccator containing silica gel beads for 1 hour under ambient pressure. Following
treatment, all samples were wrapped in aluminum foil and stored at 20 degree until
analysis. The desiccation treatment is claimed to best preserve cell components and their
Raman spectra, even though it may cause autolysis [21].
Live cells were also analyzed in order to understand photodamage in live cells and its
effect on Raman spectra. Although Raman spectra of live cells can‘t exactly be correlated
with exposure energy due to the molecular diffusion, it is possible to evaluate quantities
of the spectra. Live cells were rinsed and replaced with Tyrode’s solution in advance of
Raman measurement.
Live cells with exogenous antioxidant agents were also analyzed in order to
understand the effect of reactive oxygen species on Raman spectra and to determine
whether they could moderate the photodegradation process. 6-hydroxy-2,5,7,8-
tetramethyl-chroman-2-carboxylic acid (Trade name: Trolox) was used as an antioxidant
agent. Trolox is a model compound of water-soluble and plasma-membrane-permeable
antioxidant agent [22]. For these experiments, live cells were placed in Tyrode’s solution
with a concentration of 500 µM Trolox 30 min in advance of Raman measurements.
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Additionally, fixed cells were analyzed in order to compare living and non-living cell
samples in liquid bath media to determine the effect of cellular function on the resulting
Raman spectra. For these experiments, the cells were fixed with paraformaldehyde. Live
cells were rinsed by PBS before fixation, then placed in PBS solution at concentration of
2% paraformaldehyde for 20 min. After fixation, the cells were rinsed and immersed with
Tyrode’s buffer solution.
2.3. Evaluation of Raman band intensity
To evaluate the Raman band intensity, each Raman band was first reproduced by single-
or multi-component Lorentzian function with a linearly tilting baseline.


0
22
1
( )
n
i
i
ii
b
MultiLor xy ax
xx



  

(1)
All constants (a, bi, σi, y0, and xi) are obtained from the fitting process (i = 1,2,…,n; ‘n’ is
the number of fitting bands). Then, the Raman band intensity was defined as the integral
of a Lorentzian part of Eq. (1), that is, obtained by Eq. (2).


2
2
i
ii
b
I dx
xx





(2)
All fitting processes were performed on Igor 6.05A.
2.4. Exponential function fitting for Raman band intensity
The change in Raman band intensity is expected to correlate with a change in the number
of observable molecules, therefore is represented by a rate equation. A solution of the
equation is exponential decay or cumulative function.

0
1
( )exp
m

i
i
i
t
F tyA









(3)
τi (i = 1,2,...,m. m: the number of fitting components) is a time constant. y0 and Ai are
constants. Ai is positive for a decay component, while Ai will be negative for any
component that cumulatively rises. The fitting procedure by using Eq. (3) was applied to
the temporal change in Raman band intensity. The curve fitting procedures were
performed on Igor 6.05A.
3. Results and discussion
3.1. Change in spectra of cells depending on duration of DUV exposure
Figure 1(a) shows the obtained Raman spectra of desiccated HeLa cells with
accumulating exposure durations. Each Raman spectrum was obtained with different
durations (0.250, 0.500, 1.00, 2.50, 5.00, 7.50, 10.0, 12.5 sec) of sample exposure to the
excitation light at 16 µW/µm2 irradiation. In the spectrum obtained for the exposure
duration of 0.250 sec, several characteristic spectral peaks are observed. Table 1 shows
assignments of those characteristic peaks observed in the spectrum. All peaks are
assigned to one or more nucleotide bases and aromatic amino acids [3,4]. As the exposure
duration increases (up to 12.5 sec), the characteristic bands decay. In the spectrum
obtained for the exposure duration of 12.5 sec, the characteristic bands of nucleotide
bases and aromatic amino acids are barely visible, while two broad bands are observed at
approximately 1380 and 1610 cm1. Similar spectra have been reported as spectra of
polymeric hydrogenated amorphous carbon [23]. Polymeric hydrogenated amorphous
carbon shows two characteristic bands as D and G peaks at approximately 1400 and 1600
cm1, respectively, with a spectral dip at approximately 1500 cm1. This indicates that the
photoproduct Raman bands observed in photodegraded cells may be assigned to
polymeric amorphous carbon. We also measured the same characteristics for
photodegraded albumin (one of the model proteins), but not for the others (nucleotide
bases and lipids). The results suggest that proteins in cells were degraded into polymeric
hydrogenated amorphous carbon in DUV Raman observation.
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Fig. 1. (a) Raman spectra of desiccated cells obtained at different durations of sample
exposure (0.25, 0.5, 1, 2.5, 5, 7.5, 10, 12.5 sec) at 16 µW/µm2 irradiation. Each spectrum
is shifted vertically for clear comparison. (b-d) shows the intensity of Raman band at (b)
1483, (c) 1618, and (d) 1611 cm1 plotted over sample exposure duration, and their
corresponding fitting curves. Each spectrum was built-up from 200 different sample
positions, with the signal accumulated at the detector. This was repeated 3 times and the
error bars were set to the standard deviations between the 3 measurements.
Table 1. Raman band assignments for DUV Raman spectrum of an intact cellular
samplea
Raman shift
(cm1)
1003
1178
1211
1240
1336
G
A
Tyr
U
1358
1483
1529
1575
1618
1652
Assignment
Trp
Tyr
Tyr
T
A
A
G
C
G
A
Trp
Tyr
T
aRefer to [3,4]. The abbreviations; A: adenine, G: guanine, T: thymine, C: cytosine, U: uracil, Tyr: tyrosine,
Trp: tryptophan.
Now we focus on the exposure duration dependence of three characteristic Raman
bands at 1483 cm1 (adenine and guanine), 1618 cm1 (tryptophan and tyrosine), and 1611
cm1 (photoproduct). The intensity of these Raman bands is plotted against the sample
exposure duration, as shown in Fig. 1(b-d). The plots can clearly visualize the relationship
of the Raman band intensity and the sample exposure duration. Figure 1(b, and c) clearly
prove the Raman band intensities of purine bases and aromatic amino acids exponentially
decrease as the sample exposure duration increases up to 12.5 sec. Figure 1(d) shows that
the intensity of photoproduct Raman band cumulatively increases with an increase in the
exposure duration.
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In order to quantify the relationship between the Raman band intensity and sample
exposure duration, we applied procedures of exponential function fitting to the intensity
plots. The Raman band of purine bases at 1483 cm1 was best fit by the exponential decay
function with a single time constant. The resulting time constant of τ1 = 2.3 was obtained.
The fitting curve is shown also in Fig. 1(b). It is interesting that the degradation of two
purine bases is reproduced by a single component. The chemical structure is very similar
for adenine and guanine, and their absorbance at 244 nm is close [24]. The adenine and
guanine time profiles may represent similar reactions with excitation photons. For the
photoproduct band at 1611 cm1, the curve was best fit by the exponential cumulative
function with a single time constant of τ1 = 1.4 as also shown in Fig. 1 (d). Though the
formation of amorphous carbon photoproduct should be derived from complex pathways,
there must be a single essential pathway that dominates the growth of the photoproduct.
In contrast from the other two components, the aromatic amino acid band at 1618 cm1
was best fitted by an exponential decay function with two time constants. The fitting
curve is also shown in Fig. 1(c). The resultant time constants of (τ1, τ2) = (1.1, 0.048)
were obtained. The decay of the aromatic amino acid band showed a fast initial decay
followed by a slower decrease in intensity. Each time constant can be assigned to the
degradation of tyrosine and tryptophan. Because tyrosine and tryptophan have totally
different chemical structure, the Raman band plot was represented by two components.
We also notice that the growth of photoproduct is slower than the decays of aromatic
amino acids, but faster than the decay of purine bases. It is conceivable, although difficult
to prove, that at least one of the decay components in the aromatic amino acid band
contributes for to the rise in photoproducts. This notion is consistent with the previous
discussion that the photoproducts originate from proteins.
The degree of molecular degradation could depend on the intensity of Raman
excitation light, since the photodamage would be related to molecular phototransient
states [19], heating, and/or multiphoton absorption processes [12,18]. We compared the
molecular degradation among different Raman excitation intensities of 1.6, 4.0, 8.0, and
16 µW/µm2. Figure 2(a) shows Raman spectra obtained at 1.6 µW/µm2 irradiation for
different durations (2.50, 5.00, 10.0, 25.0, 50.0, 75.0, 100, 125 sec) of the sample
exposure. Similarly to the case of 16 µW/µm2 irradiation, the Raman intensities of
aromatic amino acids and nucleotide bases bands diminished, while the photoproduct
band rose, as the sample exposure duration increases (up to 125 sec). Figure 2(b-d) shows
plots of the intensity of the same three characteristic Raman bands at 1483, 1618, and
1611 cm1, and their fitting curves. The intensity plot of 1483 cm1 purine bases band,
1618 cm1 aromatic amino acids band, and 1611 cm1 photoproduct bands are fitted by a
single time constant exponential decay function, two time constant exponential decay
function, and a single time constant exponential cumulative function, respectively. The
fitting methods and number of time constants were the same as that for 16 µW/µm2. The
same relationships between Raman spectra of the cells and the sample exposure durations
were also observed at 4.0 and 8.0 µW/µm2 irradiation.
For a comparison of the molecular degradation among different irradiation intensities,
it is helpful to compare the obtained time constants of fitting curves. The time constants
are summarized in Table 2. For the purine bases and photoproduct, the time constants are
inversely proportional to the irradiation intensity. This is a strong indication of the fact
that the number of exposure photons determines the amount of destruction of the purine
bases and the growth of photoproduct regardless of the excitation irradiation intensity.
This implies that the photodegradation of purine bases and the growth of photoproduct is
not associated with heating, molecular phototransients, and multi-photon absorption
process in the current measurement conditions. For the aromatic amino acids band,
though the relationship between the time constants and irradiation intensity is also close
to inversely proportional, the contributions of faster and slower components are different
at different irradiation intensities. The aromatic amino acids band obtained at 16 and 8.0
µW/µm2 irradiations is mainly composed of the faster decay component, while for 1.6 and
4.0 µW/µm2 the slower component is dominant. This indicates that the degradation of
aromatic amino acids is nonlinearly dependent on the irradiation intensity and can be
associated with time-dependent effects such as heating, molecular phototransients, and
multi-photon absorption process.
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Fig. 2. (a) Raman spectra of desiccated cells obtained at different durations of sample
exposure (2.5, 5, 10, 25, 50, 75, 100, 125 sec) at 1.6 µW/µm2 irradiation. Each spectrum
is shifted vertically for clear comparison. (b-d) shows the intensity of the Raman band at
(b) 1483, (c) 1618, and (d) 1611 cm1 plotted on sample exposure duration, and their
fitting curves. The error bars were assigned in the same manner as for Fig. 1.
Table 2. Time constants obtained by the curve fitting
Irradiation
intensity
(µW/µm2)
Time constant
Purine
(1483 cm1)
2.3
Aromatic amino acid*
(1618 cm1)
1.1(0.41)
Photoproduct
(1611 cm1)
1.4
16
0.048 (0.59)
8.0
5.1
3.3 (0.38)0.11 (0.62)2.3
4.0
9.8
6.2 (0.54) 0.38 (0.46)5.5
1.6
25
14 (0.8) 0.58 (0.2)15
*In parentheses, the contribution of the component to the fitting curve is shown.
The obtained Raman spectra also contain contributions from thymine. An interesting
result was observed for the exposure duration dependence of the intensity of the thymine
Raman band at 1652 cm1. The intensity plot for 1652 cm1 thymine band is shown in Fig.
3. Interestingly, the intensity change shows the transient increase during short exposures
(up to 0.75 sec), followed by a monotonic decrease over exposures longer than 0.75 sec.
The characteristics of transient increase were observed for all different irradiation
intensities of 1.6, 4.0, 8.0, and 16 µW/µm2. The temporal behavior of the thymine band
intensity is not clearly understood, but the possible explanation of transient increase can
be either generation of transient photoproduct molecules, which show a band at the same
wavenumber or the scattering efficiency increase due to the breakdown of hypochromism,
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which is known as a phenomenon where the absorbance of nucleotide bases becomes
lower when they are stacked in DNA structure [25]. Base releases from DNA can occur
by DUV irradiation [12], resulting in the disappearance of hypochromism.

Fig. 3. The intensity of the thymine band at 1652 cm1 plotted on sample exposure
duration for 1.6 µW/µm2 irradiation and its fitting curve. The error bars were assigned in
the same manner as for Fig. 1.
3.2. Estimation of effective or allowable maximum exposure energy
The obtained fitting functions correlate the exposure duration and intensity with the
Raman band intensities. From the fitting curves, it is now possible to suggest a practically
effective maximum energy of Raman excitation light for DUV Raman observation of
cells. In terms of observation of biomolecules in cells, it is reasonable to say that the
effective maximum energy can be considered to be the exposure energy where the Raman
band intensity drops to 1/e of the intact cell signal. For a single-component exponential
decay function, the product of irradiation intensity and time constant for the fit curve
gives this exposure energy. It must be noted that values of an exponential decay function
become negligibly small with times larger than the time constant. This means that
exposure larger than the effective maximum energy just increases molecular damage in
the DUV Raman observation. For a multiple-component exponential decay function, the
same criterion can be used to derive the practically effective maximum energy.
The effective maximum energies for purine bases and aromatic amino acids at
different irradiation intensities are shown in Table 3. Regardless of irradiation intensity,
the appropriate maximum energy for observing purine bases is similar (37- 41 µJ/µm2).
For Raman band of aromatic amino acids (1618 cm1), on the other hand, the effective
maximum energy decreases by an order of magnitude with an increase of irradiation
intensity from 1.6 to 16 µW/µm2. It is concluded that observation of aromatic amino acids
must be done with smaller exposure energy for suppressing the effects of photodanage,
while the observation of purine bases doesn’t require the tuning of the excitation light
intensity.
Table 3. Effective or allowable maximum energy of excitation light exposure
Exposure energy (µJ/µm2)
Aromatic amino acid
(1618 cm1)
1.8
2.8
Irradiation intensity
(µW/µm2)
16
8.0
Purine
(1483 cm1)
37
41
Photoproduct
(1611 cm1)
22
18
4.0
1.6
39
40
9.6
18
22
24
It is also important to note the practically allowable maximum exposure energy for
suppressing the growth of photoproduct in the Raman spectra. The allowable maximum
exposure energy may be obtained using similar criterion to the effective maximum
exposure energy for observation of biomolecules, and be considered as the exposure
energy where the Raman band intensity rises to (1-1/e) of saturated photoproduct
intensity. For an exponential cumulative function, the area of the function below this
defined energy threshold is negligibly small. Thus the practically allowable maximum
energy was obtained as the product of irradiation intensity and time constant. According
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to Table 3, the practically allowable maximum energy in terms of limiting the growth of
photoproduct is therefore in the range between 18 and 24 µJ/µm2. There is little
dependence of the allowable energy on the excitation intensity.
3.3. Qualitative evaluation of photodamage on live cells
Understanding photodamage of live cells is also important, though quantification of the
molecular degradation is difficult due to molecular diffusion during exposure. Qualitative
characteristics of Raman spectra from live cells were then analyzed. Figure 4 shows the
Raman spectra of live cells obtained for different durations (5, 10, 15 sec) of sample
exposure to 16 µW/µm2 excitation light. The spectra obtained at the exposure duration of
15 s barely show the Raman band of purine bases at approximately 1480 cm1, while the
photoproduct bands at approximately 1380 and 1610 cm1 are dominantly visible. These
spectral characteristics are the same as observed in desiccated cells. Therefore, it is
concluded that the quantities of photodamage of live cells are essentially the same as in
the case of desiccated cells, although it is hard to precisely quantify the photodamage.

Fig. 4. Raman spectra of live cells in Tyrode’s solution for different durations of sample
exposure (5, 10, 15 sec). Spectra were smoothed by Loess fitting. Each spectrum is
shifted vertically for clear comparison.
Photodamage of live cells in liquid can be affected by the cellular function [26].
Living organisms show four basic responses to stressful radiation, including avoiding,
reducing, and acclimating the stress, and repairing the damage due to the stress. These
responses can be prevented by fixing the cells by paraformaldehyde. Figure 5(a) shows
the Raman spectra of fixed HeLa cells for different exposure durations (5, 10, 15 sec).
The decay of the purine base band and the growth of photoproduct in the spectra are
apparently confirmed by increasing the exposure duration. Similarly to live cells, the
spectrum for the exposure duration of 15 s barely shows the purine base band at 1480
cm1. This result indicates that cellular functions are not significantly related to the
spectral degradation. This is possibly because the cellular responses to DUV, such as
molecular repairs and movement away from the radiation, are far slower than the
observation duration.
Reactive oxygen species (ROS) play roles in molecular damage in living organisms
exposed to DUV [11]. The effect of ROS was examined using the antioxidant agent,
Trolox. Figure 5(b) shows the Raman spectra of live HeLa cells with 500 µM Trolox in
the Tyrode’s bath solution for different exposure durations (5, 10, 15 sec). The quantity of
spectral change is essentially the same as that of live cells. This indicates that ROS
doesn’t play a large role in the molecular degradation observed in DUV Raman
spectroscopy, though it should in theory mitigate some of the photodamage processes.
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Fig. 5. Raman spectra of (a) fixed cells in Tyrode’s solution, (b) live cells in Trolox
Tyrode’s solution, for different durations of sample exposure (5, 10, 15 sec). Spectra were
smoothed by Loess fitting. Each spectrum is shifted vertically for clear comparison.
4. Conclusion
In this study, we applied DUV resonant Raman spectroscopy for characterization of
molecular photodegradation in HeLa cells. Raman spectra of cells exposed to Raman
excitation light for different durations were measured and analyzed. Spectra obtained
from the cells with relatively shorter durations of exposure (0.25~2.5 sec at 16 µW/µm2)
show characteristic Raman bands of aromatic amino acids and nucleotide bases, while
longer exposure (7.5~12.5 sec at 16 µW/µm2) resulted in the disappearance of these bands
and the growth of photoproduct bands instead. We used exponential function fitting
analyses to quantify the relationship between the exposure durations and Raman
intensities of the characteristic bands. As results, the molecular degradation and the
photoproduct growth were correlated with the exposure duration. The degradation of
purine bases (1483 cm1) and the growth of photoproduct (1611 cm1) showed a linear
dependence on the number of exposure photons, while the aromatic amino acids showed a
nonlinear dependence of molecular degradation on the irradiation intensity. This implies
that only the degradation of aromatic amino acids is associated with heating, molecular
phototransients, and multi-photon absorption process.
From the obtained fitting curves, the practical maximum exposure energy for DUV
Raman observation of cell samples was suggested. The effective energy for the purine
bases (1483 cm1) was 37~41 µJ/µm2. In some cases, the photoproduct became visible
before characteristic Raman bands disappeared. The allowable energy for observation
without generating significant photoproduct (at 1611 cm1) was 18~24 µJ/µm2. The
effective exposure energy for aromatic amino acids degradation (1618 cm1) showed a
clear dependence on irradiation intensity, in contrast to other bands. 16 µW/µm2 only
allows 1.8 µJ/µm2 exposure, while 1.6 µW/µm2 allows 18 µJ/µm2, for Raman observation
of this band. This work clearly defines the allowable limits for future attempts to create
DUV resonant Raman microspectroscopy of individual components in cells excited at or
below the allowable maximum energy
Acknowledgments
This work was supported by the Japan Science and Technology (JST) organization via a
CREST project and by RIKEN via an Extreme Photonics project. The authors sincerely
acknowledge Dr. N. Hayazawa and Dr. A. Tarun of RIKEN Advanced Science Institute
for helpful comments on the manuscript and the data analyses.

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1 April 2011 / Vol. 2, No. 4 / BIOMEDICAL OPTICS EXPRESS 936